海马神经元无镁致痫性损伤对CREB磷酸化水平的影响

Effects of magnesium-free epileptiform damage on the CREB phosphorylation in hippocampal neurons

目的:观察原代培养的海马神经元经无镁人工脑脊液诱发癫痫样放电后,cAMP反应元件结合蛋白(cAMP responseelement binding protein,CREB)磷酸化表达水平的变化。方法:将所培养的神经元随机分成癫痫组和对照组,癫痫组中的神经元培养至第10天时,经无镁人工脑脊液(artificial cerebrospinal fluid,ACSF)处理3 h建立癫痫样放电模型,对照组中的神经元培养至第10天时,使用正常人工脑脊液处理3 h。使用Western blot检测p-CREB和β-actin的表达,免疫荧光双重标记磷酸化CREB(p-CREB)和神经元核抗原(neuronal nuclei,NeuN)的表达。结果:Western blot显示p-CREB水平在癫痫样放电后2 h即较对照组增强且在6 h达到高峰,在12 h和24 h都维持在增强水平,差异皆有统计学意义(P<0.01)。取癫痫样放电后6 h的神经元行免疫荧光检测,其荧光强度绝对值较对照组明显增强,差异有统计学意义(P<0.01)。结论:p-CREB水平在海马神经元无镁致痫性损伤后增强,可能在癫痫样放电中起到重要作用。

Objectlve：To observe the expression of phosphorylated cAMP response-element binding protein （p-CREB） after epilepti- form discharge of hippocampal neurons. Methods：All cultured neurons were divided into two groups：epilepsy group：on the 10th d after the incubation,maintenance medium was replaced with magnesium-free artificial cerebrospinal fluid for 3 h;control group：on the 10th d after the incubation,maintenance medium was replaced with normal artificial cerebrospinal fluid for 3 h. Changes in ex- pressions of p-CREB and β-actin were observed by Western blot. Changes in p-CREB and neuronal nuclei（NeuN） expression was investigated by double labeled immunofluorescence. Results：Results of Western blot demonstrated that p-CREB expression was increased at 2 h after epileptiform discharges in epilepsy group than in control group,peaked at 6 h and maintained increased at 12 h and 24 h. Compared with that in control group, expression of p-CREB was significantly increased in epilepsy group at each time point （P〈0.01）. Immunofluorescence intensity of p-CREB was also increased significantly in epilepsy group than in control group（P〈0.01）. Conclusions：p-CREB expression in epileptiform damage neurons is significantly enhanced,which probably closely relate with epileptiform discharge.